This proposal is directed toward understanding the molecular basis of a human metabolic disorder, hereditary fructose intolerance (HFI), its causes, incidence, distribution, and biochemical and physiological affects. This disease is caused by mutations in the liver aldolase B gene of affected individuals. Understanding these genetic issues will offer the physician and genetic counselor better tools for diagnosis and counseling of families with this potentially disastrous disease. Moreover, the biochemical analysis of mutant proteins will lead to a greater understanding of substrate specificity and structure/function relationships for this important glycolytic enzyme. The specific aims of the proposed investigations are: 1) the identification of mutations in the DNA from individuals with HFI in the American population, 2) the establishment of a national reference laboratory for the enzyme assays necessary for proper diagnosis of this and other fructose metabolic disorders from liver biopsy, and 3) the determination of the biochemical and physiological roots of this disorder by examination of the enzyme produced from the most common HFI allele. The polymerase chain reaction (PCR) will be employed to amplify DNA from blood samples of American patients for the identification of known alleles by allele specific oligonucleotide hybridization (ASO) and for the identification of any new mutant alleles. For the latter, PCR amplification of all the aldolase B encoded regions of the gene in several fragments, the identification of fragments which contain mutations by their migration in denaturing gradient gel electrophoresis (DGGE), followed by the direct sequence determination using a third primer, will be employed. A necessary complement to these molecular studies will be the determination of aldolase activity from liver biopsy. This is the best and most reliable method for diagnosis of HFI. Assays of liver biopsy will be performed on samples submitted by clinicians to confirm or rule out HFI in suspected individuals. This facility will also identify possible subjects for the molecular studies. Finally, site-directed mutagenesis will be performed to generate the mutant enzyme containing the A149P mutation, which is present in 55% of European HFI patients. Expression and purification of this mutant enzyme, followed by structural and functional analysis, will determine if this enzyme has any residual activity which may explain the normal gluconeogenesis which these patients exhibit in the absence of fructose.